Mechanism of membrane fusion by fusion protein

在本研究项目中，我们利用流感病毒融合肽研究了病毒感染的分子机制。我们特别关注于确定与膜结合的肽的结构和取向。本研究取得了以下成果。(1)用固体~ 13 P和~ 13 C NMR研究了蜂毒肽与二肉豆蔻酰磷脂酰胆碱（DMPC）双层膜结合的构象和动力学以及在脂双层系统中的磁性取向<31><13>。~ <31>1H NMR研究发现，在液晶-凝胶相变温度以上，蜂毒肽-DMPC双层膜形成了长轴平行于磁场的磁性取向的细长囊泡。<13>在<13>与脂质双层结合的13 C标记的蜂毒肽上观察13 C NMR谱。最后发现蜂毒肽是一种跨膜α-螺旋，其平均轴平行于双层法线。(2)合成了流感病毒HA 2（1-27）N端片段，并对其进行了固体核磁共振研究。结果表明，该片段的N端部分形成α-螺旋，在酸性和碱性条件下与膜的相互作用表现出不同的行为。(3)合成了流感病毒M2蛋白的跨膜片段（M2-TMP），并用~ <15>1H-N固体核磁共振谱对其进行了研究。结果表明，在DOPC和DMPC双层膜体系中，α螺旋轴分别相对于双层膜法线倾斜33°和37°。(4)用中子散射法研究了流感病毒融合肽的膜插入行为。结果表明，螺旋轴与双层膜法线成55°倾斜，螺旋位于双层膜表面附近。

In this research project, we have studied to understand the molecular mechanism of viral infectn using influenza fusion peptides. We have particularly focused to determine the structure and orientation of the peptides bund to the membrane. Following results were obtained in this research project.(1) The conformation and dynamics of melittin bound to the dimyristoylphophatidylcholin (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state ^<31>P and ^<13>C NMR spectroscopy. Using ^<31>P NMR, it was found that melittin-DMPC bilayer system forms magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature. ^<13>C NMR spectra were observed on the ^<13>C labeled melittin bound to the lipid bilayers. Finally, it was found that melittin adopts a transmembrane α-helix whose average axis is parallel to the bilayer normal.(2) N-terminal fragment of HA2 (1-27) in influenza virus was synthesized and investigated by solid state NMR spectroscopy. It was found that the N-terminal part of the fragment forms α-helix and showed different behavior of interaction with membrane at acidic pH from that at basic pH condition.(3) Transmembrane fragment of M2 protein of influenza virus (M2-TMP) was synthesized and investigated by means of ^<15>N solid state NMR spectroscopy. The results indicate that the α-helical axis is tilting by 33° and 37° from the bilayer normalin the DOPC and DMPC bilayer systems, respectively.(4) Membran insertion behavior of influenza fusion peptide was investigated by a neutron scattering method. The results indicate that the helical axis is tilting by 55° from the bilayer normal and the helix is located around the surface of bilayers.